Iron (Fe) is the most abundant metal ion found in cells, reflecting its crucial roles in the oxidation-reduction reactions upon which life depends. The rich and unique chemistry of Fe has endowed it with properties absolutely essentially for oxygen transport, ATP production and DNA synthesis. These characteristics which make Fe an obligate requirement for life also make it a potential target for preventing the growth of neoplastic cells.
In order to understand the role of Fe in cellular proliferation and the possible use of Fe chelators as effective anti-tumour agents, it is important to describe how this metal ion is transported and metabolised in normal and neoplastic cells. This is described at length in a review article entitled “Potential of Iron Chelators as Effective Anti-proliferative Agents” by D. R. Richardson which is published 1997 in Can J. Physiol. Pharmacol. 75 1164–80 and which is incorporated herein by reference.
References is also made to “Analogues of Pyridoxal Isonicotinoyl Hydrazone (PIH) as Potential Iron Chelators for the Treatment of Neoplastia” by D. R. Richardson reported at Leukaemia and Lymphoma. 1998, 31 47–60 which is also incorporated herein by reference and from which most of the following discussion has been taken.
Transportation of Fe in the serum is performed by the glycoprotein transferrin (Tf), which binds two atoms of Fe(III). Transferrin donates its Fe to cells by binding to specific Tf receptors (TfR) on the cell membrane. Upon binding to the TfR, the Tf-TfR complex is internalized within endocytotic vesicles and the Fe released from the protein by a decrease in the intravescular pH to 5.5. Apart from the specific receptor-mediated process of Fe uptake from Tf, another process consistent with non-specific adsorptive pinocytosis has also been reported in rat hepatocytes, human hepatoma cells and human melanoma cells. Once the Fe is released from Tf, it is then bound by a specific membrane transporter that remains uncharacterized. Recently, a possible candidate for this latter protein has been identified, namely the product of the gene Nramp2. This molecule has been called the divalent cation transport 1 (DCT1), and may be involved in both Fe absorption from the gut and also Fe transport across the endosomal membrane. Once Fe is transported across the membrane, it then enters a poorly characterized compartment known as the intracellular Fe pool. The identity of the pool is highly controversial and may be composed of low Mr Fe complexes of citrate, amino acids and nucleotides or alternatively, the Fe may be bound to high Mr macromolecules. Experiments have shown that the pool is composed of molecules containing Fe in the Fe(II) and Fe(III) oxidation states. In some cells, such as developing erythroid precursors, the low Mr weight Fe pool represents only a very small fraction of the total amount of Fe in the cell, whereas in other cell types, such as Chang cells, it may represent a considerably proportion of the total Fe present. Iron in the pool can be used for incorporation into Fe-containing proteins, such as the cytochromes and Fe-S proteins, and when in excess, Fe can be incorporated into the Fe storage protein ferritin.
The role played by Fe in cellular proliferation has been well demonstrated in numerous studies. For example, in the absence of Fe, ribonucleotide reductase cannot produce deoxyribonucleotides and this has a profound effect on the cell cycle resulting in a G1/S block which can lead to apoptosis. Cancer cells express very high levels of the transferring receptor (TfR), suggesting that they have a high Fe requirement. In fact, in vivo, some neoplastic cell types take up Fe from Tf at a rate that is comparable to hemoglobin producing cells such as reticulocytes. It is of interest that the host may withhold Fe during neoplastic cell proliferation, and this is found in Hodgkins and non-Hodgkins lymphoma where there is a pronounced decrease in the saturation of Tf with Fe. This latter phenomenon is known as the hypoferremic shift, which has been suggested to be a physiological response to hinder tumor cell growth. The importance of the TfR in Fe uptake and cell proliferation is demonstrated by the fact that the monoclonal antibody 42/6 which blocks the binding of Tf of the TfR, also inhibits tumor growth.
Evidence that neoplastic cells are sensitive to Fe chelation comes from work in vitro in cell culture experiments, and in vivo in clinical trials where the chelator used to treat Fe overload, desferrioxamine (DFODFO), and other Fe chelators effectively inhibit proliferation. One of the most significant reports demonstrating a pronounced therapeutic effect of DFO comes from a study done in patents with neuroblastoma (NB). In this latter trial, DFO given as an 8 hr intravenous infusion resulted in 7 of 9 patients having more than a 50% decrease in bone infiltration of tumor cells. Moreover, in 1 patient, a 48% decrease in tumor size was reported. In more recent investigations, DFO was combined with cytotoxic agents (cyclophosphamide, etoposide, thio-TEPA and carboplatin) in patients with stage III and IV NB. From 57 patients studied, there were 24 complete responses, 5 very good partial responses, 21 partial responses, 3 minor responses and 4 with progressive disease.
It has now been ascertained that DFO, which is now the drug in current clinical use, is very expensive, orally ineffective and requires long subcutaneous infusion (12–24 hr/day, 5–7 days/week) to effect significant Fe mobilization (Olivieri et al., 1997, Blood 89 739–61; Richardson et al., 1998, Am. J. Hematol. 58 299–305). The need for an orally effective and economical Fe chelator has recently been emphasized by the failure of deferiprone (also known as L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) to successfully chelate Fe from Fe-overloaded patients (Olivieri et al., 1988, New Eng. J. Med. 337 417–23). In fact, treatment of patients with this later drug resulted in hepatic fibrosis and an increase in liver Fe levels.
One important group of chelators that have shown high Fe chelation efficacy both in vitro and in vivo are those ligands of the pyridoxal isonicotinoyl hydrazone (PIH) class referred to in Richardson et al., 1998, supra. These chelators have a very high affinity and specificity for Fe(III) that is similar to that found for DFO and much greater than that of ethylenediaminetetracetic acid (EDTA) as reported in Richardson et al., 1989, supra and Vitolo et al., 1990, Inorg. Chim. Acta 733 39–50. In addition, these ligands are synthesized by a simple one-step Schiff base condensation, are economical and orally effective as discussed in Richardson et al., 1989, J. Lab Clin. Med. 131 306–15. Interestingly, PIH can chelate Fe from the mitochondrion, a site that may become loaded with Fe in the neurodegenerative disease Friedreich's ataxia (Babcock et al., 1997, Science 276 1709–12; Foury et al., 1997, FEBS Lett. 411 373–7; Rotig et al., 1997, Nature Genetics 12 215–7).
Previous studies have characterized the biological and chemical properties of analogues of PIH, some of which show higher activity on a molar basis than the parent compound itself. These compounds were derived from three groups of aromatic aldehydes, namely, pyridoxal, salicylaldehyde and 2-hydroxyl-1-naphthylaldehyde. Generally, chelators derived from pyridoxal were shown to possess high chelation efficacy but low anti-proliferative activity, while ligands derived from 2-hydroxy-1-naphthylaldehyde had high Fe chelation efficacy and potent anti-proliferative activity. Hence, aroylhydrazones derived from pyridoxal were considered to be possibly useful as agents to treat Fe overload disease while chelators derived from 2-hydroxyl-1-naphthylaldehyde were considered to have better potential for the treatment of cancer. It should be noted that many other Fe chelators have also demonstrated anti-proliferative activity, including DFO. In fact, some of the most potent effects of DFO have been reported when this drug was used against the pediatric tumor neuroblastoma. In Cory et al., 1995, Adv. Enzyme Regul. 35 55–68 and Liu et al., 1995, Prog. Med. Chem. 32 1–35, there are disclosed a closely related group of chelators derived from 2-pyridylcarboxaldehyde and thiosemicarbazide (e.g. 3-aminopyridine-2-carboxaldehyde thiosemicarbazone) which were found to be among the most effective inhibitors of ribonucleotide reductase yet identified. However, these chelators, while having high anti-proliferative properties, were found to have only moderate chelation efficacy and moderate lipophilicity which made such chelators less efficient in regard to treatment of Fe overload diseases.
Friedreich's ataxia (FA) is a severe neurodegenerative condition. In 97% of patients the disease is due to a GAA triplet repeat expansion in intron 1 of the FRDA gene resulting in a marked decrease in its expression. The protein encoded by this gene is known as frataxin and is found within the mitochondrion. Over the last few years evidence has accumulated to suggest that frataxin plays a role in mitochondrial Fe metabolism. Studies using the yeast cell showed that deletion of the homologous gene (YFH1), resulted in an accumulation of mitochondrial Fe resulting in the loss of mitochondrial DNA, [Fe-S] cluster-containing enzymes, and respiration. Like the human FRDA gene, YFH1 encodes a mitochondrial protein (Yfh1p). When YFH1 was reintroduced back into the yeast, mitochondrial Fe was exported back out into the cytosol, suggesting a “mitochondrial Fe cycle”.
Consistent with the knockout yeast model, it was noted that reductions in mitochondrial DNA, complex I, complex II/III, and aconitase occurred in the heart of FA patients, observations consistent with mitochondrial damage. In addition, it was reported increased Fe deposition in the heart, liver, and spleen was reported in FA patients in a pattern consistent with a mitochondrial location. This work suggesting the pathology of FA in humans is caused by mitochondrial Fe overload was strongly supported by work showing Fe deposits within the heart myofibrils, defective myocardial and skeletal muscle mitochondrial respiration, and perturbations in the heme biosynthesis pathway.
Since the pathology of FA is linked to mitochondrial Fe overload, new therapies based on these results could provide hope for FA patients. One strategy is the use of specific Fe chelators that can permeate the mitochondrion. Already a trial supported by the National Institute of Health is investigating the use of the clinically used Fe chelator desferrioxamine (DFO) to treat FA patients. However, DFO cannot efficiently mobilize Fe from cells, and previous studies have demonstrated that it is not effective at mobilizing Fe from Fe-loaded mitochondria in reticulocytes.
In contrast to DFO, another chelator known as pyridoxal isonicotinoyl hydrazone (PIH) shows high activity at mobilising Fe from an experimental model of mitochondrial Fe overload in reticulocytes. A variety of studies, in vitro, in vivo, and a clinical trial, have demonstrated that PIH and its analogues show potential for the treatment of Fe-overload disease.
Although a lot of work has been done to develop Fe chelators for use in medical applications, there is still a need for new chelators which have safe and efficacious characteristics. The present inventors have now developed new Fe chelators that have been found as suitable candidate for use in treating Fe overload disease. The present inventors have synthesized a new group of ligands known as 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) analogues. Several PCIH analogues are more active than DFO or PIH at mobilizing Fe from a neuroepithelioma cell line (SK-N-MC), and showed low anti-proliferative activity.